The present invention relates to a method of dwarfing plants and a molecule used in said method.
Miniaturizing plants is important from various aspects of agriculture and horticulture. For example, miniaturizing the plant height or the culm length can produce ornamental plants with new aesthetic values, and miniaturizing vegetables or fruits can create crops having new commercial values such as being bite-sized. Aside from these industrial applications, miniaturizing experimental plants will not only make them easy to handle but will also reduce the space necessary for culturing the plants, which leads to efficient use of the laboratory space.
In plant miniaturization, the characteristic that reduces the plant height or the culm length compared to the wild type (the normal type) is called dwarfism. A number of strains and varieties of rice having this characteristic have been produced by xcex3-ray or chemical mutagenesis, and they have been genetically analyzed for a long time. Over 60 different genes have so far been found to be involved in dwarfism (Rice Genetic Newsletter, 12: 30-32 (1995)). However, little is known about why the dwarf phenotype is expressed, except that a few dwarf mutants are caused by mutations at the genes involved in the biosynthesis of gibberellin, which is a plant growth hormone.
Among the dwarf mutants of rice, the Daikoku type dwarfism is known for suppressing the elongation of the second internode, thereby reducing the plant height to less than one half of the normal, and rendering the grain small and circular (Masao Akemine, Nihon Gakujutsukai Houkoku (Japan Science Council Report), 1: 108-314 (1925), Tsutsumu Nakayama, Shinshudai Bunrigakubu Kiyou (Journal of Faculty of Liveral Arts and Science Shinshu University), 4: 1-31 (1954); Man-emon Takahashi and Kazuyoshi Takeda, Hokudai Nou Houbun Kiyou (Memories of the Faculty of Agriculture Hokkaido University), 7: 32-43 (1969)). Furthermore, genetic analysis has clarified that the Daikoku type dwarfism is regulated by a single recessive gene, d1, on rice chromosome 5 (Nagao and Takahashi, J. Fac. Agr., Hokkaido Univ. 53: 72-130 (1963); Iwata and Omura, Jpn. J. Genet. 51: 135-137 (1976)). In d1 gene mutants, the plant hormone gibberellin contents are high enough to be equal to the normal type, and the plant height cannot be recovered to normal by exogenous gibberellin treatments (Suge, H. and Y. Murakami, Plant and Cell Physiol. 9: 411-414 (1968)). This characteristic suggests that the d1 gene is not involved in the biosynthesis of gibberellin within the plant body, but is presumedly involved in a genetic locus associated with the gibberellin receptor molecule or the signal transduction thereafter (Mitsunaga, S., Tashiro, T. and Yamaguchi, J., Theor. Appl. Genet. 87: 705-712 (1994)).
Therefore, it is important to isolate the Daikoku type dwarfism gene d1 and to reveal the function of its gene product not only to clarify the morphogenesis in plants but also to artificially control the plant sizes utilizing the knowledge obtained.
An objective of the present invention is to isolate the d1 gene which is involved in dwarfism of plants. Another objective of the present invention is to provide a method of dwarfing plants by controlling the expression of the d1 gene and to provide a molecule to control the expression of the d1 gene.
Rice dwarfism gene d1 has so far been known to encode a protein associated with dwarfism of rice and to exist at a certain locus in the vast region of rice chromosome 5. In order to isolate the d1 gene from the vast chromosomal region, the present inventors determined the location of the d1 gene using a linkage analysis and succeeded in isolating the desired gene by the map-based cloning method. More specifically, the region on the chromosome where the d1 gene is located was narrowed to a region between specific molecular markers by linkage analysis. A physical map of the area around the region thus narrowed was then made by aligned YAC clones. As a result of EST mapping, the inventors succeeded in identifying a single cDNA clone xe2x80x9cST5933xe2x80x9d on these YAC clones as the candidate for the d1 gene.
The nucleotide sequence of isolated clone ST5933 (362 base pairs) was analyzed by a homology search for the gene sequence in the database. This gene was characterized as the xcex1 subunit of a G protein in plants. However, this gene has not previously been associated with plant dwarfism thus far.
G proteins are known to function as the receptors for hormones or the factors involved in signal transduction from the receptors in living organisms including humans. Rice dwarfism gene d1 may also be a receptor for the plant hormone gibberellin or be involved in the signal transduction thereafter. G protein genes that have functions similar to those described above are expected to exist not only in rice plants but also widely in the plant kingdom. The present inventors have found that a wide variety of plants can be dwarfed by repressing the expression of the d1 gene or its homologous genes in other plants.
Thus, the present invention relates to a method of dwarfing plants by repressing the expression of the d1 gene or its homologues, which is associated with the dwarfism of plants, and to a molecule used to repress the expression of the genes. More specifically, it relates to
(1) a DNA for dwarfing plants encoding an antisense RNA complementary to the transcription product of the DNA of (a), (b), or (c) below:
(a) a DNA encoding a protein comprising the amino acid sequence described in SEQ ID NO: 1;
(b) a DNA comprising the nucleotide sequence described in SEQ ID NO: 2; or
(c) a DNA which hybridizes with the DNA comprising the nucleotide sequence described in SEQ ID NO: 2, and encodes a protein functionally equivalent to a protein comprising the amino acid sequence described in SEQ ID NO: 1;
(d) a DNA encoding the amino acid sequence of SEQ ID NO: 1 comprising one or more conservative amino acid substitutions
(2) a DNA for dwarfing plants encoding an RNA having ribozyme activity to specifically cleave the transcription product of the DNA described in (a), (b), or (c) of (1);
(3) a DNA for dwarfing plants encoding an RNA which represses the expression of the DNA described in (a), (b), or (c) of (1) in the plant cell through a co-repressive effect;
(4) use of the DNA of (1), (2), or (3) for dwarfing plants;
(5) a method of dwarfing plants, which comprises repressing the expression of the DNA described in (a), (b), or (c) of (1) in a plant cell;
(6) the method of (5), wherein the expression of the DNA described in (a), (b), or (c) of (1) is repressed by expressing the DNA of (1), (2), or (3) in the plant cell;
(7) a method of dwarfing plants, which comprises:
(a) introducing a substance that represses the expression of the DNA described in (a), (b), or (c) of (1) into the plant cells; and
(b) regenerating said plant cells to obtain transgenic plants;
(8) The method of (7), wherein said substance is the DNA of (1), (2), or (3);
(9) a transformed plant cell retaining the DNA of (1), (2), or (3) and capable of expressing said DNA;
(10) a dwarf transgenic plant containing the cell of (9); and
(11) a reproductive media of the plant of (10).